Kidney injury molecule-1 (KIM-1) is the most upregulated protein in proximal tubular epithelial cells in various states characterized by epithelial cell dedifferentiation: ischemia, toxic renal injury, and renal cell carcinoma. We have cloned, generated cells and animals expressing wild-type and mutant KIM-1, as well as monoclonal and polyclonal antibodies to human, mouse, rat, pig, dog, and zebrafish KIM-1. The KIM-1 ectodomain is cleaved and found in the urine of patients with acute kidney injury (AKI) and chronic kidney disease (CKD), serving as a sensitive and specific kidney injury biomarker of injury, qualified by the FDA for preclinical safety studies, and currently used in many clinical safety studies. We have discovered that KIM-1 transforms kidney epithelial cells into semiprofessional phagocytes making it the first nonmyeloid phosphatidylserine receptor. We have described a novel KIM-1-mediated phagocytosis pathway by which autophagy regulates phagocytosis and MHC-restricted antigen presentation in epithelial cells. While KIM-1 expression in AKI is adaptive, a transgenic mouse which expresses KIM-1 in the renal tubule develops CKD with severe fibrosis, secondary hypertension, and cardiac hypertrophy. A mutant mouse lacking the extracellular mucin domain, important for phagocytosis, is protected against development of fibrosis. We have found that KIM-1 internalizes albumin-bound fatty acids and oxidized lipoproteins which induce a DNA-repair response (DDR) and trigger profibrotic factor production. We have demonstrated that the DDR leading to G2/M cell cycle arrest is an important contributor to a senescence?associated profibrotic secretory phenotype. The goal of this proposal is to further characterize the functional role of KIM-1 during chronic kidney injury. We hypothesize that persistent KIM-1 signaling activates a proliferative response as well as a DDR leading to G2/M arrest and a prosecretory fibrotic phenotype. KIM-1-ligand interactions lead to altered cell-matrix interactions leading to enhanced TGF? activation. Finally with persistent KIM-1 expression a state of autophagy insufficiency is generated resulting in GATA activation and an additional non-DDR dependent fibrotic response. In Specific Aim 1 we will evaluate whether KIM-1-induced mTOR activation leads to dedifferentiation and proliferation which, together with G2/M arrest due to the DDR, results in a profibrotic secretory phenotype. In Specific Aim 2 we will evaluate whether the upregulation of ECM components by KIM-1 expression promotes activation of TGF? and YAP-induced entry into the cell cycle which, in the setting of the DDR, potentiates kidney fibrosis. In Specific Aim 3 we will determine whether an impaired autophagic response associated with uptake of endocytic ligands by KIM-1 triggers the activation of GATA4, DNA damage, senescence and G2/M arrest. There are therapeutic implications for our studies since agents that interfere with mTOR, fibronectin, or the HIPPO-YAP pathway or stimulate autophagy, are in development and our studies will hopefully motivate KIM-1-directed therapeutics to prevent or mitigate progression of CKD.